The present invention relates to peptides which are suited to inhibit HPV-E6 proteins, DNAs coding for them and the use of both, particularly for eliminating HPV-positive cells, e.g. HPV tumor cells.
Human papilloma viruses (HPVs) are closely related with the development of carcinomas. The involvement of HPVs in the development of the cervical carcinoma is well characterized, and various findings refer to the fact that HPVs play a causal part in the etiology of this carcinoma. On a molecular level, sequences of HPVs, particularly HPV 16 and 18, are detectable in about 95% of the cervical carcinoma biopsies. In this connection, the HPV DNA is usually present in integrated form in the genome of the tumor cells. It frequently comprises deletions and/or rearrangements, which never refer to the early HPV genes, namely the E6 and E7 genes. These genes code for the HPV proteins E6 and E7 which are responsible for the formation and manifestation of HPV carcinomas. In this connection, the HPV proteins have a synergistic effect. On the other hand, there are references to the effect that they also display opposite activities. For example, the E7 protein induces apoptosis, whereas the E6 protein inhibits it. This is due to the fact that the E6 protein binds to p53 directly or indirectly, i.e. via ubiquintin-protein ligase E6-AP, and inhibits it, so that p53 can no longer display its apoptosis-inducing activity. The E6 protein also has an anti-apoptotic activity independent of p53. The genes of the E6 and E7 proteins are expressed via polycistronic mRNAs, the transcription being controlled by a common promoter. Experiments of inhibiting the latter and/or E6-/E7 mRNAs are known. These experiments resulted in a parallel inhibition of the growth of HPV tumor cells. However, elimination of such cells, which is most desired, could not be achieved by this.
Therefore, it is the object of the present invention to provide a product by which HPV tumor cells can be eliminated.
According to the invention this is achieved by the subject matters defined in the claims.
The applicant found that HPV-E6 proteins bind to short peptides. He screened a randomized oligopeptide library which comprises randomly generated peptide sequences with a xe2x80x9cpeptide aptamer systemxe2x80x9d in which the HPV-E6 protein was used as a screening sample. He found that short peptides, particularly those listed in Table 1, bind HPV-E6 proteins. He also found that these peptides are suited to inhibit activities of HPV-E6 proteins, e.g. their anti-apoptotic activity. In addition, he observed that an elimination of HPV-positive cells, particularly HPV tumor cells, can be achieved by this inhibition.
According to the invention, the applicant""s insights are used to provide a peptide which is selected from the peptides listed in Table 1, wherein the peptide may comprise a sequence modification of up to 40%, particularly 20% and most particularly 10%.
Peptides according to the invention are suited to bind HPV-E6 proteins and inhibit them as regards their activities, e.g. as regards their anti-apoptotic activity.
The expression xe2x80x9cHPV-E6 proteinsxe2x80x9d comprises an E6 protein of any HPV type, particularly of HPV1, 5, 6, 11, 16, 18, 31, 33 or 35. An E6 protein can have a wild-type sequence or a sequence differing therefrom by one or several amino acids. Furthermore, it may be present in shortened form, i.e. it is only available as the fragment which is necessary to bind to p53, ubiquintin-protein ligase E6-AP or another binding participant of the E6 protein. The fragment can also be present in multiple copies within a polypeptide molecule. An E6 protein or a fragment thereof can also be present in the form of a fusion protein.
Peptides according to the invention can be provided by common methods in which peptides are tested as regards their binding to HPV-E6 proteins. Such methods are e.g. the xe2x80x9cpeptide aptamerxe2x80x9d or xe2x80x9cbacteriophage displayxe2x80x9d method. It is favorable to use the xe2x80x9cpeptide aptamerxe2x80x9d method which is described in the examples and which is a modification of the above-mentioned method.
Peptides according to the invention can be present as such or in combination with other substances, e.g. (poly)peptides. The combination may consist in linking the peptides according to the invention with the (poly)peptides via linkers, e.g. disulfide bridges. The peptides according to the invention can also be fused with the (poly)peptides, so that the peptides according to the invention are present in the form of fusion (poly)peptides. For example, leader peptides, such as penetratin from Drosophila antennapedia or VP22 from HSV1, which support the absorption of the peptides according to the invention in cells, offer themselves as (poly)peptides for fusion (poly)peptides. On the other hand, polypeptides which are linked with the peptide according to the invention via linkers, can be e.g. carrier proteins, such as transferrin, which are not considered foreign by the body. Several peptides according to the invention can also be simultaneously present in combination with an above-mentioned substance.
A further subject matter of the present invention relates to a nucleic acid, particularly a DNA which codes for a peptide according to the invention. Such a DNA can be present in vectors, particularly expression vectors. A person skilled in the art is familiar with examples thereof. In the case of an expression vector for E. coli, these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8. For the expression in yeast, e.g. pY100 and Ycpad1 have to be mentioned while e.g. pKCR, PEFBOS, cDM8 or pCEV4 have to be indicated for the expression in animal cells. The baculovirus expression vector pAcSGHisNT-A is especially suited for the expression in insect cells. It is also possible to use viruses, e.g. adenovirus, vaccinia virus, adeno-associated virus (AAV) or retroviruses, such as MoMuLV, HaMuSV, MuMTV, RSV or GaIV, for the expression in animal cells.
The person skilled in the art is familiar with suitable cells to express a DNA according to the invention, which is present in an expression vector. Examples of such cells comprise the E. coli strains HB101, DH1, x1776, JM101, JM109, BL21 and SG13009, the yeast strain Saccharomyces cerevisiae and the animal cells L, NIH 3T3, FM3A, CHO, COS, Vero and HeLa as well as the insect cells sf9.
The person skilled in the art knows in which way the DNA according to the invention has to be inserted in an expression vector. He is also familiar with the fact that this DNA can be inserted in combination with a DNA coding for another peptide or polypeptide, so that the DNA according to the invention can be expressed in the form of a fusion polypeptide.
In addition, the person skilled in the art knows conditions of culturing transformed or transfected cells. He is also familiar with methods of isolating and purifying the peptide or fusion polypeptide, which is expressed by the DNA according to the invention.
A further subject matter of the present invention relates to an antibody directed against an above peptide or fusion polypeptide. Such an antibody can be prepared by common methods. It may be polyclonal or monoclonal. For its preparation it is favorable to immunize animalsxe2x80x94particularly rabbits or chickens for a polyclonal antibody and mice for a monoclonal antibodyxe2x80x94with an above (fusion) polypeptide or with fragments thereof. Further xe2x80x9cboostersxe2x80x9d of the animals can be effected with the same (fusion) polypeptide or with fragments thereof. The polyclonal antibody may then be obtained from the animal serum or egg. For the preparation of the monoclonal antibody, animal spleen cells are isolated and fused with myeloma cells.
A further subject matter of the present invention relates to a pharmaceutical composition which contains one or several peptides according to the invention and/or DNAs encoding them as well as conventional auxiliary agents. The auxiliary agents used can be e.g. carriers, binders, blasting agents, lubricants, solvents, solution aids, release accelerators, release retarders, emulsifiers, stabilizers, etc. Such a composition can be used as usual, e.g. orally or parenterally. The suitable dosage is determined in the individual case as usual.
A further subject matter of the present invention relates to a diagnostic composition which contains one or several peptides according to the invention. HPV-E6 proteins can be detected by such a composition. This can be used to detect HPV-associated diseases, such as HPV infections, HPV dysplasias of HPV carcinomas. Such a detection comprises e.g. (a) collection of a cell sample from a patient, (b) contacting of the cell sample with a peptide according to the invention under conditions permitting the specific binding of the peptide to an HPV-E6 protein, and (c) detection of the peptide. This detection can be made by standard methods. For example, the peptides according to the invention can be present in the liquid phase or be bound to a solid carrier and be labeled in different ways. The person skilled in the art is familiar with suitable markers and labeling methods. The peptides can also be detected by antibodies according to the invention. The latter are also suited to control the therapeutic course of an HPV-associated disease treated with peptides according to the invention.
By means of the present invention it is possible to bind HPV-E6 proteins and inhibit their activities, particularly their anti-apoptotic activity. This serves for achieving the elimination of HPV-positive cells, particularly HPV tumor cells. HPV-positive cells can also be diagnosed. Therefore, the present invention is suitable to take diagnostic and therapeutic steps against HPV-associated diseases, particularly HPV infections, HPV dysplasias or HPV carcinomas. In addition, the peptides according to the invention and/or DNAs encoding them represent a basis for the development of fully new active substances against the above diseases.